Polyadenylation is the process of eukaryotic mRNA processing in which 3′ end cleavage occurs, followed by the addition of as many as 250 adenosine residues. Messenger RNA polyadenylation is important for cellular processes including transcription termination, splicing, mRNA transport, translation, and mRNA stability. Polyadenylation requires at least five protein complexes, including the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), two cleavage factors (CFI and CFII), and the poly (A) polymerase. Other factors, including the poly(A)-binding protein II (which mediates poly7(A) tail length), the U1A small nuclear ribonucleoprotein (SnRNP) (which interacts with both CPSF and the poly(A)polymerase) and DSEF-1 (which binds G-rich auxiliary elements), also contribute to efficient polyadenylation.
In the past, it was believed that efficient polyadenylation required the sequence AAUAAA near the 3′ end. More recent studies have brought this belief into question. Computer-aided surveys of sequences available in GenBank and other online datasets have suggested that the incidence of AAUAAA in mRNA 3′ ends of certain tissues is far lower than previously suspected (reviewed in MacDonald et al., “Reexamining the Polyadenylation Signal: Were We Wrong About AAUAAA?” Mol. Cell. Endocrinol. 190:1–8 (2002)). The lower incidence of AAUAAA is especially notable in mRNAs from male germ cells of several mammalian species (Meijer et al., “Molecular Characterization of the Testis Specific c-abl mRNA in Mouse,” EMBO J. 6:4041–4048 (1987); Oppi et al., “Nucleotide Sequence of Testis-Derived c-abl cDNAs: Implications for Testis-Specific Transcription and abl Oncogene Activation,” Proc. Natl. Acad. Sci. USA 84:8200–8204 (1987); Øyen et al., “Subunits of Cyclic Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase Show Differential and Distinct Expression Patterns During Germ Cell Differentiation: Alternative Polyadenylation in Germ Cells Gives Rise to Unique Smaller-Sized mRNA Species,” Biol. Reprod. 43:46–54 (1990); Wallace et al., “Two Distinct Forms of the 64,000 Mr Protein of the Cleavage Stimulation Factor are Expressed in Mouse Male Germ Cells,” Proc. Natl. Acad. Sci. USA 96:6763–6768 (1999)). Moreover, there have been a number of reports of alternative polyadenylation in germ cells, in which one site is used in most somatic tissues, but a different site is used in germ cells (Meijer et al., “Molecular Characterization of the Testis Specific c-abl mRNA in Mouse,” EMBO J. 6:4041–4048 (1987); Oppi et al., “Nucleotide Sequence of Testis-Derived c-abl cDNAs: Implications for Testis-Specific Transcription and abl Oncogene Activation,” Proc. Natl. Acad. Sci. USA 84:8200–8204 (1987); Foulkes et al., “Pituitary Hormone FSH Directs the CREM Functional Switch During Spermatogenesis,” Nature 362:264–267 (1993); Ravnik et al., “The Developmentally Restricted Pattern of Expression in the Male Germ Line of a Murine Cyclin A, Cyclin A2, Suggests Roles in Both Mitotic and Meiotic Cell Cycles,” Dev. Biol. 173:69–78 (1996); Edwalds-Gilbert et al., “Alternative Poly(A) Site Selection in Complex Transcription Units: Means to an End?” Nucl. Acids Res. 25:2547–2561 (1997)). Together, these data argue strongly for a modified polyadenylation mechanism in male germ cells.
In studying AAUAAA-independent polyadenylation in mice, it was determined that there were two distinct forms of the essential polyadenylation protein CstF-64 (Cleavage stimulation Factor, 64,000 Mr) in male germ cells (Wallace et al., “Two Distinct Forms of the 64,000 Mr Protein of the Cleavage Stimulation Factor are Expressed in Mouse Male Germ Cells,” Proc. Natl. Acad. Sci. USA 96:6763–6768 (1999)). One form of CstF-64 was expressed in nuclei of cells in every tissue examined, and is referred to as the somatic CstF-64. This protein was expressed from a gene on the X chromosome in both mice (gene designation Cstf2) and humans (CSTF2). The other form was found only in male germ cells and brain, and is referred to as the variant CstF-64, or τCstF-64 (Wallace et al., “Two Distinct Forms of the 64,000 Mr Protein of the Cleavage Stimulation Factor are Expressed in Mouse Male Germ Cells,” Proc. Natl. Acad. Sci. USA 96:6763–6768 (1999); Dass et al., “The Gene for a Variant Form of the Polyadenylation Protein CstF-64 is on Chromosome 19 and is Expressed in Pachytene Spermatocytes in Mice,” J. Biol. Chem. 276:8044–8050 (2001)). Because genes on the X and Y chromosomes are inactivated during male meiosis (Monesi, V., “Differential Rate of Ribonucleic Acid Synthesis in the Autosomes and Sex Chromosomes During Male Meiosis in the Mouse,” Chromosoma 17:11–21 (1965); McCarrey et al., “Human Testis-Specific PGK Gene Lacks Introns and Possesses Characteristics of a Processed Gene,” Nature 326:501–504 (1987); Handel et al., “Role of Sex Chromosomes in the Control of Male Germ-Cell Differentiation,” Ann. NY Acad. Sci. 637:64–73 (1991); McCarrey et al., “Semiquantitative Analysis of X-Linked Gene Expression During Spermatogenesis in the Mouse: Ethidium-Bromide Staining of RT-PCR Products,” Genetics Analysis Technology and Applications 9:117–123 (1992)), it was proposed that the somatic CstF-64 was inactivated during pachytene of male meiosis due to sequestration of the X and Y chromosomes within the sex body (Wallace et al., “Two Distinct Forms of the 64,000 Mr Protein of the Cleavage Stimulation Factor are Expressed in Mouse Male Germ Cells,” Proc. Natl. Acad. Sci. USA 96:6763–6768 (1999)), and that τCstF-64 was a paralogous gene expressed from an autosome.
It seemed likely that this phenomenon was true not only of rodents (Wallace et al., “Two Distinct Forms of the 64,000 Mr Protein of the Cleavage Stimulation Factor are Expressed in Mouse Male Germ Cells,” Proc. Natl. Acad. Sci. USA 96:6763–6768 (1999), but of all eutherian mammals (Handel et al., “Role of Sex Chromosomes in the Control of Male Germ-Cell Differentiation,” Ann. NY Acad. Sci. 637:64–73 (1991)), suggesting the presence and potential importance of τCstF-64 in human spermatogenesis.
The further identification and characterization of these RNA polyadenylation proteins in mammals and the genes that encode them is now needed to provide a greater understanding of the mechanisms underlying RNA processing in specialized cells, including germ cells, and to provide diagnostic tools and therapeutic treatment for the disorders related to the absence, or improper functioning, of these genes and the proteins or polypeptides they encode.
The present invention is directed at overcoming these and other deficiencies in the art.